A novel way of amino acid-specific assignment in1H-15N HSQC spectra with a wheat germ cell-free protein synthesis system

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Abstract
For high-throughput protein structural analyses, it is indispensable to develop a reliable protein overexpression system. Although many protein overexpression systems, such as ones utilizing E. coli cells, have been developed, a lot of proteins functioning in solution still were synthesized as insoluble forms. Recently, a novel wheat germ cell-free protein synthesis system was developed, and many of such proteins were synthesized as soluble forms. This means that the applicability of this protein synthesis method to determination of the functional structures of soluble proteins. In our previous work, we synthesized 15N-labeled proteins with this wheat germ cell-free system, and confirmed this applicability on the basis of the strong similarity between the 1H-15N HSQC spectra for native proteins and the corresponding ones for synthesized ones. In this study, we developed a convenient and reliable method for amino acid selective assignment in 1H-15N HSQC spectra of proteins, using several inhibitors for transaminases and glutamine synthase in the process of protein synthesis. Amino acid selective assignment in 1H-15N HSQC spectra is a powerful means to monitor the features of proteins, such as folding, intermolecular interactions and so on. This is also the first direct experimental evidence of the presence of active transaminases and glutamine synthase in wheat germ extracts. Abbreviation: HSQC - heteronuclear single quantum coherence spectroscopy